Optimal Use of Treatment Modalities and Treatment Results of Osteosarcoma and Soft Tissue Sarcomas

Soft tissue sarcomas are malignant tumours of mesenchymal origin. Because of infiltrative growth pattern, simple enucleation of the tumour causes a high rate of local recurrence. Instead, these tumours should be resected with a rim of normal tissue around the tumour. Data on the adequate margin width are scarce. At Helsinki University Central Hospital (HUCH) a multidisciplinary treatment group started in 1987. Surgical resection with a wide margin (2.5 cm) is the primary aim. In case of narrower margin radiation therapy is necessary. The role of adjuvant chemotherapy remains unclear. Our aims were to study local control by the surgical margin and to develop a new prognostic tool to aid decision-making on which patients should receive adjuvant chemotherapy. Patients with soft tissue sarcoma of the extremity or the trunk wall referred to HUCH during 1987-2002 form material in Studies I and II. External validation material comes from the Lund university sarcoma registry. The smallest surgical margin of at least 2.5 centimetres yielded local control of 89 per cent at five years. Amputation rate was 9 per cent. The proposed prognostic model with necrosis, vascular invasion, size on a continuous scale, depth, location and grade worked well both in Helsinki material and in the validation material, and it also showed good calibration. Based on the present study, we recommend the smallest surgical margin of 2-3 centimetres in soft tissue sarcoma irrespective of grade. Improvement in local control was present but modest in margins wider than 1 centimetre. In cases where gaining a wider margin would lead to a considerable loss of function, smaller margin is to be considered combined to radiation therapy. Patients treated with inadequate margins should be offered radiation therapy irrespective of tumour grade. Our new prognostic model to estimate 10-year survival probability in patients with soft tissue sarcoma of the extremities or trunk wall showed good dicscrimination and calibration. For time being the prognostic model is available for scientific use and further validations. In the future, the model may aid in clinical decision-making. For operable osteosarcoma, neoadjuvant multidrug chemotherapy followed by delayed surgery and multidrug adjuvant chemotherapy is the treatment of choice. Overall survival rates at five years are approximately 75 per cent in modern trials with classical osteosarcoma. All patients diagnosed and reported to the Finnish Cancer Registry with osteosarcoma in Finland during 1971-2005 form the material in Studies III and IV. Limb-salvage rate increased from 23 per cent to 78 per cent during 1971-2005. The 10-year sarcoma-specific survival for the whole study population improved from 32 per cent to 62 per cent. It was 75 per cent for patients with a local high-grade osteosarcoma of the extremity diagnosed during 1991-2005. This study outlines the improved prognosis of osteosarcoma patients in Finland with modern chemotherapy. The 10-year survival rates are good also in an international scale. Nonetheless, their limb-salvage rate remains inferior to those seen for highly selected patient series. Overall, the centralisation of osteosarcoma treatment would most likely improve both survival and limb-salvage rates even further.

Smoking patterns and lung function : A longitudinal twin study

In many countries, the prevalence of smoking and smokers average cigarette consumption have decreased, with occasional smoking and daily light smoking (1-4 cigarettes per day, CPD) becoming more common. Despite these changes in smoking patterns, the prevalence of chronic obstructive pulmonary disease (COPD), a disorder characterized by a progressive decline in lung function, continues to rise globally. Smoking is the most important factor causing COPD, however, not all smokers develop the disease. Genetic factors partly explain the inter-individual differences in lung function and susceptibility of some smokers to COPD. No earlier research on the genetic and environmental determinants of lung function or on the phenomenon of light smoking exists in the Finnish population. Further, the association between low-rate smoking patterns and COPD remains partly unknown. This thesis aimed to study the prevalence and consistency of light smoking longitudinally in the Finnish population, to assess the characteristics of light smokers, and to examine the risks of chronic bronchitis and COPD associated with changing smoking patterns over time. A further aim was to estimate longitudinally the proportions of genetic and environmental factors that explain the inter-individual variances in lung function. Data from the Older Finnish Twin Cohort, including same-sex twin pairs born in Finland before 1958, were used. Smoking patterns and chronic bronchitis symptoms were consistently assessed in surveys conducted in 1975, 1981, and 1990. National registry data on reimbursement eligibilities and medication purchases were used to define COPD. Lung function data were obtained from a subsample of the cohort, 217 female twin pairs, who attended spirometry in 2000 and 2003 as part of the Finnish Twin Study on Ageing. The genetic and environmental influences on lung function were estimated by using genetic modeling. This thesis found that light smokers are more often female, well-educated, and exhibit a healthier lifestyle than heavy smokers. At individual level, light smoking is rarely a constant pattern. Light smoking, reducing from heavier smoking to light smoking, and relapsing to light smoking after quitting, are among patterns associated with an increased risk of chronic bronchitis and COPD. Constant light smoking is associated with an increased use of inhaled anticholinergics, a medication for CODP. In addition to smoking, other environmental factors influence lung function in the older age. During a three-year follow-up, new environmental effects influencing spirometry values were observed, whereas the genes affecting lung function remained mostly the same. In conclusion, no safe level of daily smoking exists with regard to pulmonary diseases. Even daily light smoking in middle-age is associated with increased respiratory morbidity later in life. Smoking reduction does not decrease the risk of COPD, and should not be recommended as an alternative to quitting smoking. In elderly people, attention should also be drawn to other factors that can prevent poor lung function.

NK-and T-cell function in chronic myeloid leukemia patients treated with interferon-? monotherapy

Chronic myeloid leukemia (CML) is one of the most studied human malignancies. It is caused by an autonomously active tyrosine kinase BCR-ABL, which is a result from a translocation between chromosomes 9 and 22 in the hematopoietic stem cell. As an outcome, a Philadelphia (Ph) chromosome is formed. BCR-ABL causes disturbed cell proliferation among other things. Although targeted tyrosine kinase inhibitor therapy has been developed in the beginning of the millenium and the survival rate has increased significantly, it is still not known why some patients benefit more from the treatment than others. Furthermore, the therapy is not considered to be curative. Before the era of tyrosine kinase inhibitors, the first-line treatment for CML was interferon-? (IFN-?). However, only a small proportion of patients benefitted from the treatment. Of these patients, a few were able to discontinue the treatment without renewal of the disease. The mechanism of IFN-? is not completely understood, but it is believed that differences in the immune system can be one of the reasons why some patients have better therapy response. Kreutzman, Rohon et al. have recently discovered that patients who have been able to stop IFN-? treatment have an increased number of NK- and T-cells. They also have a unique clonal T-cell population and more cytotoxic CD8+ T-cells and less CD4+ T-cells. The aim of this master’s thesis was to study the function of T- and NK-cells in IFN-? treated patients. Although it was shown earlier that IFN-? treated patients have increased NK-cell count, the function of these cells was unknown. Therefore, we have now investigated the killing potential of patients’ NK-cells, their activation status and cell surface antigen expression. In addition, we have also studied the activation status of patients’ T-cells and their cytotoxic properties. We observed that NK-cells from patients treated with IFN-? are unable to kill leukemic cells (K562) than NK-cells from healthy controls. In addition, patients on IFN-? treatment have more active T-cells and their NK-cells have an undifferentiated immunoregulatory phenotype. Patients that have been able to stop the treatment have anergic T-and NK-cells. As a conclusion our results suggest that IFN-? therapy induces increased NK-cell count, NK-cell immunoregulatory functions and more active T-cells. After stopping IFN-? therapy, NK- and T-cells from CML patients restore anergy typical for CML.

The function and regulation of the U11-48K protein in U12-dependent splicing

The removal of noncoding sequences, or introns, from the eukaryotic messenger RNA precursors is catalyzed by a ribonucleoprotein complex known as the spliceosome. In most eukaryotes, two distinct classes of introns exist, each removed by a specific type of spliceosome. The major, U2-type introns account for over 99 % of all introns, and are almost ubiquitous. The minor, U12-type introns are found in most but not all eukaryotes, and reside in conserved locations in a specific set of genes. Due to their slow excision rates, the U12-type introns are expected to be involved in the regulation of the genes containing them by inhibiting the maturation of the messenger RNAs. However, little information is currently available on how the activity of the U12-dependent spliceosome itself is regulated. The levels of many known splicing factors are regulated through unproductive alternative splicing events, which lead to inclusion of premature STOP codons, targeting the transcripts for destruction by the nonsense-mediated decay pathway. These alternative splice sites are typically found in highly conserved sequence elements, which also contain binding sites for factors regulating the activation of the splice sites. Often, the activation is achieved by binding of products of the gene in question, resulting in negative feedback loops. In this study, I show that U11-48K, a protein factor specific to the minor spliceosome, specifically recognizes the U12-type 5' splice site sequence, and is essential for proper function of the minor spliceosome. Furthermore, the expression of U11-48K is regulated through a feedback mechanism, which functions through conserved sequence elements that activate alternative splicing and nonsense-mediated decay. This mechanism is conserved from plants to animals, highlighting both the importance and early origin of this mechanism in regulating splicing factors. I also show that the feedback regulation of U11-48K is counteracted by a component of the major spliceosome, the U1 small nuclear ribonucleoprotein particle, as well as members of the hnRNP F/H protein family. These results thus suggest that the feedback mechanism is finely tuned by multiple factors to achieve precise control of the activity of the U12-dependent spliceosome.